Reshma Ramracheya

Voltage-Gated Ion Channels in Human Pancreatic β-Cells: Electrophysiological Characterization and Role in Insulin Secretion

OBJECTIVE— To characterize the voltage-gated ion channels in human β-cells from nondiabetic donors and their role in glucose-stimulated insulin release. RESEARCH DESIGN AND METHODS— Insulin release was measured from intact islets. Whole-cell patch-clamp experiments and measurements of cell capacitance were performed on isolated β-cells. The ion channel complement was determined by quantitative PCR. RESULTS— Human β-cells express two types of voltage-gated K+ currents that flow through delayed rectifying (KV2.1/2.2) and large-conductance Ca2+-activated K+ (BK) channels. Blockade of BK channels (using iberiotoxin) increased action potential amplitude and enhanced insulin secretion by 70%, whereas inhibition of KV2.1/2.2 (with stromatoxin) was without stimulatory effect on electrical activity and secretion. Voltage-gated tetrodotoxin (TTX)-sensitive Na+ currents (NaV1.6/1.7) contribute to the upstroke of action potentials. Inhibition of Na+ currents with TTX reduced glucose-stimulated (6–20 mmol/l) insulin secretion by 55–70%. Human β-cells are equipped with L- (CaV1.3), P/Q- (CaV2.1), and T- (CaV3.2), but not N- or R-type Ca2+ channels. Blockade of L-type channels abolished glucose-stimulated insulin release, while inhibition of T- and P/Q-type Ca2+ channels reduced glucose-induced (6 mmol/l) secretion by 60–70%. Membrane potential recordings suggest that L- and T-type Ca2+ channels participate in action potential generation. Blockade of P/Q-type Ca2+ channels suppressed exocytosis (measured as an increase in cell capacitance) by >80%, whereas inhibition of L-type Ca2+ channels only had a minor effect. CONCLUSIONS— Voltage-gated T-type and L-type Ca2+ channels as well as Na+ channels participate in glucose-stimulated electrical activity and insulin secretion. Ca2+-activated BK channels are required for rapid membrane repolarization. Exocytosis of insulin-containing granules is principally triggered by Ca2+ influx through P/Q-type Ca2+ channels.

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans.

Glucagon, secreted from pancreatic islet α cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring β cells, or to an intrinsic glucose sensing by the α cells themselves. We examined hormone secretion and Ca2+ responses of α and β cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn2+ signalling was blocked, but was reversed by low concentrations (1–20 μM) of the ATP-sensitive K+ (KATP) channel opener diazoxide, which had no effect on insulin release or β cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 μM). Higher diazoxide concentrations (≥30 μM) decreased glucagon and insulin secretion, and α- and β-cell Ca2+ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 μM) stimulated glucagon secretion, whereas high concentrations (>10 μM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na+ (TTX) and N-type Ca2+ channels (ω-conotoxin), but not L-type Ca2+ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca2+ channels and α-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an α-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

Regulation of PKD by the MAPK p38δ in Insulin Secretion and Glucose Homeostasis

Summary Dysfunction and loss of insulin-producing pancreatic β cells represent hallmarks of diabetes mellitus. Here, we show that mice lacking the mitogen-activated protein kinase (MAPK) p38δ display improved glucose tolerance due to enhanced insulin secretion from pancreatic β cells. Deletion of p38δ results in pronounced activation of protein kinase D (PKD), the latter of which we have identified as a pivotal regulator of stimulated insulin exocytosis. p38δ catalyzes an inhibitory phosphorylation of PKD1, thereby attenuating stimulated insulin secretion. In addition, p38δ null mice are protected against high-fat-feeding-induced insulin resistance and oxidative stress-mediated β cell failure. Inhibition of PKD1 reverses enhanced insulin secretion from p38δ-deficient islets and glucose tolerance in p38δ null mice as well as their susceptibility to oxidative stress. In conclusion, the p38δ-PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic β cells, pointing to a pivotal role for this pathway in the development of overt diabetes mellitus.

GLP-1 Inhibits and Adrenaline Stimulates Glucagon Release by Differential Modulation of N- and L-Type Ca2+ Channel-Dependent Exocytosis

Glucagon secretion is inhibited by glucagon-like peptide-1 (GLP-1) and stimulated by adrenaline. These opposing effects on glucagon secretion are mimicked by low (1-10 nM) and high (10 muM) concentrations of forskolin, respectively. The expression of GLP-1 receptors in alpha cells is <0.2% of that in beta cells. The GLP-1-induced suppression of glucagon secretion is PKA dependent, is glucose independent, and does not involve paracrine effects mediated by insulin or somatostatin. GLP-1 is without much effect on alpha cell electrical activity but selectively inhibits N-type Ca(2+) channels and exocytosis. Adrenaline stimulates alpha cell electrical activity, increases [Ca(2+)](i), enhances L-type Ca(2+) channel activity, and accelerates exocytosis. The stimulatory effect is partially PKA independent and reduced in Epac2-deficient islets. We propose that GLP-1 inhibits glucagon secretion by PKA-dependent inhibition of the N-type Ca(2+) channels via a small increase in intracellular cAMP ([cAMP](i)). Adrenaline stimulates L-type Ca(2+) channel-dependent exocytosis by activation of the low-affinity cAMP sensor Epac2 via a large increase in [cAMP](i).

Reduced Insulin Exocytosis in Human Pancreatic β-Cells With Gene Variants Linked to Type 2 Diabetes

The majority of genetic risk variants for type 2 diabetes (T2D) affect insulin secretion, but the mechanisms through which they influence pancreatic islet function remain largely unknown. We functionally characterized human islets to determine secretory, biophysical, and ultrastructural features in relation to genetic risk profiles in diabetic and nondiabetic donors. Islets from donors with T2D exhibited impaired insulin secretion, which was more pronounced in lean than obese diabetic donors. We assessed the impact of 14 disease susceptibility variants on measures of glucose sensing, exocytosis, and structure. Variants near TCF7L2 and ADRA2A were associated with reduced glucose-induced insulin secretion, whereas susceptibility variants near ADRA2A, KCNJ11, KCNQ1, and TCF7L2 were associated with reduced depolarization-evoked insulin exocytosis. KCNQ1, ADRA2A, KCNJ11, HHEX/IDE, and SLC2A2 variants affected granule docking. We combined our results to create a novel genetic risk score for β-cell dysfunction that includes aberrant granule docking, decreased Ca2+ sensitivity of exocytosis, and reduced insulin release. Individuals with a high risk score displayed an impaired response to intravenous glucose and deteriorating insulin secretion over time. Our results underscore the importance of defects in β-cell exocytosis in T2D and demonstrate the potential of cellular phenotypic characterization in the elucidation of complex genetic disorders.

γ-Aminobutyric Acid (GABA) Is an Autocrine Excitatory Transmitter in Human Pancreatic β-Cells

OBJECTIVE Paracrine signaling via γ-aminobutyric acid (GABA) and GABAA receptors (GABAARs) has been documented in rodent islets. Here we have studied the importance of GABAergic signaling in human pancreatic islets. RESEARCH DESIGN AND METHODS Expression of GABAARs in islet cells was investigated by quantitative PCR, immunohistochemistry, and patch-clamp experiments. Hormone release was measured from intact islets. GABA release was monitored by whole-cell patch-clamp measurements after adenoviral expression of α1β1 GABAAR subunits. The subcellular localization of GABA was explored by electron microscopy. The effects of GABA on electrical activity were determined by perforated patch whole-cell recordings. RESULTS PCR analysis detected relatively high levels of the mRNAs encoding GABAAR α2, β3, γ2, and π subunits in human islets. Patch-clamp experiments revealed expression of GABAAR Cl− channels in 52% of β-cells (current density 9 pA/pF), 91% of δ-cells (current density 148 pA/pF), and 6% of α-cells (current density 2 pA/pF). Expression of GABAAR subunits in islet cells was confirmed by immunohistochemistry. β-Cells secreted GABA both by glucose-dependent exocytosis of insulin-containing granules and by a glucose-independent mechanism. The GABAAR antagonist SR95531 inhibited insulin secretion elicited by 6 mmol/l glucose. Application of GABA depolarized β-cells and stimulated action potential firing in β-cells exposed to glucose. CONCLUSIONS Signaling via GABA and GABAAR constitutes an autocrine positive feedback loop in human β-cells. The presence of GABAAR in non–β-cells suggests that GABA may also be involved in the regulation of somatostatin and glucagon secretion.

Chronic Palmitate Exposure Inhibits Insulin Secretion by Dissociation of Ca2+ Channels from Secretory Granules

Summary Long-term (72 hr) exposure of pancreatic islets to palmitate inhibited glucose-induced insulin secretion by >50% with first- and second-phase secretion being equally suppressed. This inhibition correlated with the selective impairment of exocytosis evoked by brief (action potential-like) depolarizations, whereas that evoked by long (∼250 ms) stimuli was unaffected. Under normal conditions, Ca2+ influx elicited by brief membrane depolarizations increases [Ca2+]i to high levels within discrete microdomains and triggers the exocytosis of closely associated insulin granules. We found that these domains of localized Ca2+ entry become dispersed by long-term (72 hr), but not by acute (2 hr), exposure to palmitate. Importantly, the release competence of the granules was not affected by palmitate. Thus, the location rather than the magnitude of the Ca2+ increase determines its capacity to evoke exocytosis. In both mouse and human islets, the palmitate-induced secretion defect was reversed when the β cell action potential was pharmacologically prolonged.

Regulation of glucagon secretion by glucose: paracrine, intrinsic or both?

Glucagon secretion is regulated by glucose but the mechanisms involved remain hotly debated. Both intrinsic (within the α‐cell itself) and paracrine (mediated by factors released β‐ and/or δ‐cells) have been postulated. Glucagon secretion is maximally suppressed by glucose concentrations that do not affect insulin and somatostatin secretion, a finding that highlights the significance of intrinsic regulation of glucagon secretion. Experiments on islets from mice lacking functional ATP‐sensitive potassium channels (KATP‐channels) indicate that these channels are critical to the α‐cells capacity to sense changes in extracellular glucose. Here, we review recent data on the intrinsic and paracrine regulation of glucagon secretion in human pancreatic islets. We propose that glucose‐induced closure of the KATP‐channels, via membrane depolarization, culminates in reduced electrical activity and glucagon secretion by voltage‐dependent inactivation of the ion channels involved in action potential firing. We further demonstrate that glucagon secretion measured in islets isolated from donors with type‐2 diabetes is reduced at low glucose and that glucose stimulates rather than inhibits secretion in these islets. We finally discuss the relative significance of paracrine and intrinsic regulation in the fed and fasted states and propose a unifying model for the regulation of glucagon secretion that incorporates both modes of control.

Role of KATP Channels in Glucose-Regulated Glucagon Secretion and Impaired Counterregulation in Type 2 Diabetes

Summary Glucagon, secreted by pancreatic islet α cells, is the principal hyperglycemic hormone. In diabetes, glucagon secretion is not suppressed at high glucose, exacerbating the consequences of insufficient insulin secretion, and is inadequate at low glucose, potentially leading to fatal hypoglycemia. The causal mechanisms remain unknown. Here we show that α cell KATP-channel activity is very low under hypoglycemic conditions and that hyperglycemia, via elevated intracellular ATP/ADP, leads to complete inhibition. This produces membrane depolarization and voltage-dependent inactivation of the Na+ channels involved in action potential firing that, via reduced action potential height and Ca2+ entry, suppresses glucagon secretion. Maneuvers that increase KATP channel activity, such as metabolic inhibition, mimic the glucagon secretory defects associated with diabetes. Low concentrations of the KATP channel blocker tolbutamide partially restore glucose-regulated glucagon secretion in islets from type 2 diabetic organ donors. These data suggest that impaired metabolic control of the KATP channels underlies the defective glucose regulation of glucagon secretion in type 2 diabetes.

Reversible changes in pancreatic islet structure and function produced by elevated blood glucose

Diabetes is characterized by hyperglycaemia due to impaired insulin secretion and aberrant glucagon secretion resulting from changes in pancreatic islet cell function and/or mass. The extent to which hyperglycaemia per se underlies these alterations remains poorly understood. Here we show that β-cell-specific expression of a human activating KATP channel mutation in adult mice leads to rapid diabetes and marked alterations in islet morphology, ultrastructure and gene expression. Chronic hyperglycaemia is associated with a dramatic reduction in insulin-positive cells and an increase in glucagon-positive cells in islets, without alterations in cell turnover. Furthermore, some β-cells begin expressing glucagon, whilst retaining many β-cell characteristics. Hyperglycaemia, rather than KATP channel activation, underlies these changes, as they are prevented by insulin therapy and fully reversed by sulphonylureas. Our data suggest that many changes in islet structure and function associated with diabetes are attributable to hyperglycaemia alone and are reversed when blood glucose is normalized.